Pathophysiology Of Cystic Fibrosis Lung Disease Research Articles

Lung Inflammatory Genes in Cystic Fibrosis and Their Relevance to Cystic Fibrosis Transmembrane Conductance Regulator Modulator Therapies.

Cystic fibrosis (CF) is a monogenic syndrome determined by over 2000 mutations in the CF Transmembrane Conductance Regulator (CFTR) gene harbored on chromosome 7. In people with CF (PWCF), lung disease is the major determinant of morbidity and mortality and is characterized by a clinical phenotype which differs in the presence of equal mutational assets, indicating that genetic and environmental modifiers play an important role in this variability. Airway inflammation determines the pathophysiology of CF lung disease (CFLD) both at its onset and progression. In this narrative review, we aim to depict the inflammatory process in CF lung, with a particular emphasis on those genetic polymorphisms that could modify the clinical outcome of the respiratory disease in PWCF. The natural history of CF has been changed since the introduction of CFTR modulator therapies in the clinical arena. However, also in this case, there is a patient-to-patient variable response. We provide an overview on inflammatory/immunity gene variants that affect CFLD severity and an appraisal of the effects of CFTR modulator therapies on the inflammatory process in lung disease and how this knowledge may advance the optimization of the management of PWCF.

Update on Calcium Signaling in Cystic Fibrosis Lung Disease

Cystic fibrosis (CF) is an autosomal recessive disorder characterized by mutations in the cystic fibrosis transmembrane conductance regulator gene, which causes multifunctional defects that preferentially affect the airways. Abnormal viscosity of mucus secretions, persistent pathogen infections, hyperinflammation, and lung tissue damage compose the classical pathological manifestation referred to as CF lung disease. Among the multifunctional defects associated with defective CFTR, increasing evidence supports the relevant role of perturbed calcium (Ca2+) signaling in the pathophysiology of CF lung disease. The Ca2+ ion is a critical player in cell functioning and survival. Its intracellular homeostasis is maintained by a fine balance between channels, transporters, and exchangers, mediating the influx and efflux of the ion across the plasma membrane and the intracellular organelles. An abnormal Ca2+ profile has been observed in CF cells, including airway epithelial and immune cells, with heavy repercussions on cell function, viability, and susceptibility to pathogens, contributing to proinflammatory overstimulation, organelle dysfunction, oxidative stress, and excessive cytokines release in CF lung. This review discusses the role of Ca2+ signaling in CF and how its dysregulation in airway epithelial and immune cells contributes to hyperinflammation in the CF lung. Finally, we provide an outlook on the therapeutic options that target the Ca2+ signaling to treat the CF lung disease.

Mining GWAS and eQTL data for CF lung disease modifiers by gene expression imputation.

Genome wide association studies (GWAS) have identified several genomic loci with candidate modifiers of cystic fibrosis (CF) lung disease, but only a small proportion of the expected genetic contribution is accounted for at these loci. We leveraged expression data from CF cohorts, and Genotype-Tissue Expression (GTEx) reference data sets from multiple human tissues to generate predictive models, which were used to impute transcriptional regulation from genetic variance in our GWAS population. The imputed gene expression was tested for association with CF lung disease severity. By comparing and combining results from alternative approaches, we identified 379 candidate modifier genes. We delved into 52 modifier candidates that showed consensus between approaches, and 28 of them were near known GWAS loci. A number of these genes are implicated in the pathophysiology of CF lung disease (e.g., immunity, infection, inflammation, HLA pathways, glycosylation, and mucociliary clearance) and the CFTR protein biology (e.g., cytoskeleton, microtubule, mitochondrial function, lipid metabolism, endoplasmic reticulum/Golgi, and ubiquitination). Gene set enrichment results are consistent with current knowledge of CF lung disease pathogenesis. HLA Class II genes on chr6, and CEP72, EXOC3, and TPPP near the GWAS peak on chr5 are most consistently associated with CF lung disease severity across the tissues tested. The results help to prioritize genes in the GWAS regions, predict direction of gene expression regulation, and identify new candidate modifiers throughout the genome for potential therapeutic development.

Non-coding RNA in cystic fibrosis

Non-coding RNAs (ncRNAs) are an abundant class of RNAs that include small ncRNAs, long non-coding RNAs (lncRNA) and pseudogenes. The human ncRNA atlas includes thousands of these specialised RNA molecules that are further subcategorised based on their size or function. Two of the more well-known and widely studied ncRNA species are microRNAs (miRNAs) and lncRNAs. These are regulatory RNAs and their altered expression has been implicated in the pathogenesis of a variety of human diseases. Failure to express a functional cystic fibrosis (CF) transmembrane receptor (CFTR) chloride ion channel in epithelial cells underpins CF. Secondary to the CFTR defect, it is known that other pathways can be altered and these may contribute to the pathophysiology of CF lung disease in particular. For example, quantitative alterations in expression of some ncRNAs are associated with CF. In recent years, there has been a series of published studies exploring ncRNA expression and function in CF. The majority have focussed principally on miRNAs, with just a handful of reports to date on lncRNAs. The present study reviews what is currently known about ncRNA expression and function in CF, and discusses the possibility of applying this knowledge to the clinical management of CF in the near future.

Airway Mucosal Host Defense Is Key to Genomic Regulation of Cystic Fibrosis Lung Disease Severity.

The severity of cystic fibrosis (CF) lung disease varies widely, even for Phe508del homozygotes. Heritability studies show that more than 50% of the variability reflects non-cystic fibrosis transmembrane conductance regulator (CFTR) genetic variation; however, the full extent of the pertinent genetic variation is not known. We sought to identify novel CF disease-modifying mechanisms using an integrated approach based on analyzing "in vivo" CF airway epithelial gene expression complemented with genome-wide association study (GWAS) data. Nasal mucosal RNA from 134 patients with CF was used for RNA sequencing. We tested for associations of transcriptomic (gene expression) data with a quantitative phenotype of CF lung disease severity. Pathway analysis of CF GWAS data (n = 5,659 patients) was performed to identify novel pathways and assess the concordance of genomic and transcriptomic data. Association of gene expression with previously identified CF GWAS risk alleles was also tested. Significant evidence of heritable gene expression was identified. Gene expression pathways relevant to airway mucosal host defense were significantly associated with CF lung disease severity, including viral infection, inflammation/inflammatory signaling, lipid metabolism, apoptosis, ion transport, Phe508del CFTR processing, and innate immune responses, including HLA (human leukocyte antigen) genes. Ion transport and CFTR processing pathways, as well as HLA genes, were identified across differential gene expression and GWAS signals. Transcriptomic analyses of CF airway epithelia, coupled to genomic (GWAS) analyses, highlight the role of heritable host defense variation in determining the pathophysiology of CF lung disease. The identification of these pathways provides opportunities to pursue targeted interventions to improve CF lung health.

Regulation of mRNA turnover in cystic fibrosis lung disease.

Cystic fibrosis (CF) is an autosomal recessive disease due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, F508del-CFTR being the most frequent mutation. The CF lung is characterized by a hyperinflammatory phenotype and is regulated by multiple factors that coordinate its pathophysiology. In CF the expression of CFTR as well as proinflammatory genes are regulated at the level of messenger RNA (mRNA) stability, which subsequently affect translation. These mechanisms are mediated by inflammatory RNA-binding proteins as well as small endogenous noncoding microRNAs, in coordination with cellular signaling pathways. These regulatory factors exhibit altered expression and function in vivo in the CF lung, and play a key role in the pathophysiology of CF lung disease. In this review, we have described the role of mRNA stability and associated regulatory mechanisms in CF lung disease. WIREs RNA 2017, 8:e1408. doi: 10.1002/wrna.1408 For further resources related to this article, please visit the WIREs website.

MicroRNA Expression in Cystic Fibrosis Airway Epithelium.

MicroRNAs (miRs) have emerged as major regulators of the protein content of a cell. In the most part, miRs negatively regulate target mRNA expression, with sets of miRs predicted to regulate certain signaling pathways. The miR expression profile of endobronchial brushings is altered in people with cystic fibrosis (CF) compared to those without CF. How this impacts on CF has important implications for our growing understanding of the pathophysiology of CF lung disease and the development of new therapeutics to treat its pulmonary manifestations. Herein we discuss the potential consequences of altered miR expression in CF airway epithelium particularly with respect to cystic fibrosis transmembrane conductance regulator (CFTR) expression, innate immunity and toll-like receptor signalling and explore how best to exploit these changes for therapeutic benefit.

Relative contribution of Prevotella intermedia and Pseudomonas aeruginosa to lung pathology in airways of patients with cystic fibrosis

BackgroundPatients with cystic fibrosis (CF) with Pseudomonas aeruginosa lung infections produce endobronchial mucus plugs allowing growth of obligate anaerobes including Prevotella spp. Whether obligate anaerobes contribute to the pathophysiology of...

Association of airway cathepsin B and S with inflammation in cystic fibrosis

Irreversible tissue damage within the cystic fibrosis (CF) lung is mediated by proteolytic enzymes during an inflammatory response. Serine proteinases, in particular neutrophil elastase (NE), have been implicated however, members of the cysteine proteinase family may also be involved. The aim of this study was to determine cathepsin B and S levels in cystic fibrosis (CF) sputum and to assess any relationship to recognized markers of inflammation such as sputum NE, interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-alpha), urine TNF receptor 1 (TNFr1), plasma IL-6, and serum C-reactive protein (CRP). Proteinase activities were measured in the sputum of 36 clinically stable CF patients using spectrophotometric and fluorogenic assays. Immunoblots were also used to confirm enzyme activity data. All other parameters were measured by ELISA. Patients had a mean age of 27.2 (8.2) years, FEV. of 1.6 (0.79) L and BMI of 20.7 (2.8). Both cathepsin B and S activities were detected in all samples, with mean concentrations of 18.0 (13.5) microg/ml and 1.6 (0.88) microg/ml, respectively and were found to correlate not only with each other but with NE, TNF-alpha and IL-8 (in all cases . < 0.05). Airway cathepsin B further correlated with circulatory IL-6 and CRP however, no relationship for either cathepsin was observed with urine TNFr1. This data indicates that cathepsin B and S may have important roles in the pathophysiology of CF lung disease and could have potential as markers of inflammation in future studies.

Adhesion to and biofilm formation on IB3-1 bronchial cells by Stenotrophomonas maltophilia isolates from cystic fibrosis patients

BackgroundStenotrophomonas maltophilia has recently gained considerable attention as an important emerging pathogen in cystic fibrosis (CF) patients. However, the role of this microorganism in the pathophysiology of CF lung disease remains largely unexplored. In the present study for the first time we assessed the ability of S. maltophilia CF isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line. The role of flagella on the adhesiveness of S. maltophilia to IB3-1 cell monolayers was also assessed by using fliI mutant derivative strains.ResultsAll S. maltophilia CF isolates tested in the present study were able, although at different levels, to adhere to and form biofilm on IB3-1 cell monolayers. Scanning electron and confocal microscopy revealed S. maltophilia structures typical of biofilm formation on bronchial IB3-1 cells. The loss of flagella significantly (P < 0.001) decreased bacterial adhesiveness, if compared to that of their parental flagellated strains. S. maltophilia CF isolates were also able to invade IB3-1 cells, albeit at a very low level (internalization rate ranged from 0.01 to 4.94%). Pre-exposure of IB3-1 cells to P. aeruginosa PAO1 significantly increased S. maltophilia adhesiveness. Further, the presence of S. maltophilia negatively influenced P. aeruginosa PAO1 adhesiveness.ConclusionsThe main contribution of the present study is the finding that S. maltophilia is able to form biofilm on and invade CF-derived IB3-1 bronchial epithelial cells, thus posing a rationale for the persistence and the systemic spread of this opportunistic pathogen in CF patients. Experiments using in vivo models which more closely mimic CF pulmonary tissues will certainly be needed to validate the relevance of our results.

Mapping the Lung Proteome in Cystic Fibrosis

The pathophysiology of cystic fibrosis (CF) lung disease remains incompletely understood. Novel mechanisms in the pathogenesis of CF lung disease may be discovered by studying the patterns of protein expression in bronchoalveolar lavage fluid (BALF). We used shotgun proteomics to analyze BALF samples from 8 CF and 4 control subjects. Differential protein expression between CF and control subjects was determined using spectral counting and statistical analysis. Using Gene Ontology analysis, we identified enriched biological modules and then applied network analysis to construct a protein interaction map in CF lung disease. Shotgun proteomics analysis of BALF identified hundreds of proteins whose differential enrichment or depletion robustly distinguished the CF phenotype from normal controls. Functional categorization and network analysis identified key processes, including the immune response and proteolytic activity that are known contributors to CF lung disease. Importantly, this approach also implicated abnormalities in previously unsuspected pathways, such as dysregulation of the complement system that may have critical roles in the pathogenesis of CF lung disease. By integrating shotgun proteomics with statistical and computational analyses, we have developed a promising approach to understand the pathophysiology of CF lung disease. Our approach should be applicable to a wide range of proteomics-based clinical research.

Cystic Fibrosis: Evolution from a Fatal Disease of Infancy with a Clear Phenotype to a Chronic Disease of Adulthood with Diverse Manifestations

Cystic fibrosis (CF) is the most common life-threatening genetic condition in the Caucasian population. Since the complete pathologic description of the disease in 1938, tremendous progress has been made in identifying its genetic basis, the pathophysiology of CF lung disease, and the development of new therapies. This issue of CRAI will review several areas of progress in our understanding and treatment of CF.

Cystic Fibrosis Transmembrane Conductance Regulator-independent Phagosomal Acidification in Macrophages

It was reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) is required for acidification of phagosomes in alveolar macrophages (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Here we determined whether the CFTR chloride channel is a generalized pathway for chloride entry into phagosomes in macrophages and whether mutations in CFTR could contribute to alveolar macrophage dysfunction. The pH of mature phagolysosomes in macrophages was measured by fluorescence ratio imaging using a zymosan conjugate containing Oregon Green(R) 488 and tetramethylrhodamine. Acidification of phagolysosomes in J774A.1 macrophages (pH approximately 5.1 at 45 min), murine alveolar macrophages (pH approximately 5.3), and human alveolar macrophages (pH approximately 5.3) was insensitive to CFTR inhibition by the thiazolidinone CFTR(inh)-172. Acidification of phagolysosomes in alveolar macrophages isolated from mice homozygous for DeltaF508-CFTR, the most common mutation in cystic fibrosis, was not different compared with that in alveolar macrophages isolated from wild-type mice. We also measured the kinetics of phagosomal acidification in J774A.1 and murine alveolar macrophages using a zymosan conjugate containing fluorescein and tetramethylrhodamine. Phagosomal acidification began within 3 min of zymosan binding and was complete within approximately 15 min of internalization. The rate of phagosomal acidification in J774A.1 cells was not slowed by CFTR(inh)-172 and was not different in alveolar macrophages from wild-type versus DeltaF508-CFTR mice. Our data indicate that phagolysosomal acidification in macrophages is not dependent on CFTR channel activity and do not support a proposed mechanism for cystic fibrosis lung disease involving defective phagosomal acidification and bacterial killing in alveolar macrophages.

Quality, Size, and Composition of Pediatric Endobronchial Biopsies in Cystic Fibrosis

Studies on airway remodeling in children with cystic fibrosis (CF) may be hampered by difficulty in obtaining evaluable endobronchial biopsy specimens because of large amounts of mucus and inflammation in the CF airway. We prospectively assessed how the quality of biopsy specimens obtained from children with CF compare with those from children with other airway diseases. Fiberoptic bronchoscopy with endobronchial biopsy was performed in 67 CF children (age range, 0.2 to 16.8 years), 34 children with wheeze/asthma (W/A), and 64 control children with chronic respiratory symptoms. Up to three biopsy specimens were taken and stained with hematoxylin and eosin. Biopsy specimen size and structural composition were quantified using stereology. At least one evaluable biopsy specimen was obtained in 72% of CF children, in 79% of children with W/A, and in 72% of control subjects (difference was not significant). The use of large biopsy forceps (2.0 mm) rather than small biopsy forceps (1.0 mm) [odds ratio (OR), 5.8; 95% confidence interval (CI), 1.1 to 29.8; p = 0.037] and the number of biopsy specimens taken (odds ratio, 2.6; 95% confidence interval, 1.3 to 5.2; p = 0.006) significantly contributed to the success rate. Biopsy size and composition were similar between groups, except that CF children and those patients with W/A had a higher percentage of the biopsy specimen composed of muscle than did control subjects (median 6.2% and 9.7% vs 0.9%, respectively; p = 0.002). Biopsy size and quality are adequate for the study of airway remodeling in CF children as young as 2 months of age. Researchers should use large forceps when possible and take at least two biopsy specimens per patient. An increased airway smooth muscle content of the airway mucosa may contribute to the pathophysiology of CF lung disease.

Cystic fibrosis and airway submucosal glands

The chronic pulmonary infections and inflammation associated with cystic fibrosis (CF) are responsible for almost all the morbidity and mortality of this disease. Our understanding of the mechanisms that underlie the very early stages of CF lung disease, that result directly from mutations in the CF gene, is relatively poor. However, the demonstration that the predominant sites of expression of the CF gene in normal lungs are the submucosal glands, together with the histological observations showing that hyperplasia of these glands and mucin occlusion of the gland ducts are the earliest signs of disease in the CF lung, suggest that malfunction of the submucosal glands may be an important factor contributing to the early pathophysiology of CF lung disease. This review describes the function of submucosal glands in normal lungs, and the way in which their function may be disrupted in CF and may thus contribute to the early stages of CF lung disease.

Altered terminal glycosylation and the pathophysiology of CF lung disease

Altered terminal glycosylation, with increased fucosylation and decreased sialylation, is a hallmark of the cystic fibrosis (CF) glycosylation phenotype. The glycosylation phenotype of CF airway epithelial cells has been modulated by the expression of wtCFTR. Understanding the effects of mutant CFTR on glycosylation may provide further insight into the regulation of glycoconjugate processing as well as new approaches to the therapy of CF.

Modifier genes in cystic fibrosis lung disease

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene and is characterized by progressive bronchiectatic lung disease and pancreatic exocrine insufficiency. A broad spectrum of disease severity exists; some individuals with CF die early in childhood, whereas others live well into adulthood with only mild lung disease. It is now clear that CFTR genotype alone does not account for the wide diversity in CF pulmonary phenotype. Evidence is accumulating that secondary genetic factors separate from the CFTR locus significantly influence the severity of CF lung disease. The general classes of these potential modifier genes include inflammatory and antiinflammatory mediators, antioxidants, mediators of airway reactivity, molecules involved in CFTR trafficking, and alternative ion channels. The best-studied CF candidate modifiers include mannose-binding lectin, glutathione-S-transferase, transforming growth factor-β1, tumor necrosis factor-α, β2-adrenegic receptor, and HLA class II antigens. Ongoing studies designed to identify genetic modifiers of CF pulmonary phenotype may offer new insights into the pathophysiology of CF lung disease and provide leads for new CF therapeutic interventions. (J Lab Clin Med 2003;141:237-41)

Airway surface pH in subjects with cystic fibrosis.

The cystic fibrosis (CF) transmembrane conductance regulator protein can transport bicarbonate and may therefore regulate airway surface (AS) pH. Disturbances of AS pH could contribute to the pathophysiology of CF lung disease. Five studies were carried out including the following: study 1) nasal pH measurements were made in 25 CF and 10 non-CF adults using an antimony pH probe. Mean nasal pH was significantly lower in the CF group. Nasal potential difference may have been a confounding factor; study 2) in a fresh cohort of CF and non-CF subjects, no significant difference was found between the two groups using a gold pH probe; study 3) simultaneous nasal pH measurements were made in 15 CF and 15 non-CF adults using both probes. In the CF group, there was a trend for the antimony probe to read lower than the gold probe. In the non-CF group, the antimony probe read higher. The pH difference noted in study 1 related to technical factors; study 4) the effect of acute changes in serum acid/base balance on nasal pH was assessed in five non-CF adults. Nasal pH was not altered by either acute respiratory acidosis or alkalosis; study 5) nasal and lower airway pH was measured in five CF and six non-CF children. No difference was found between the groups. There was a correlation between nasal and lower airway pH. The authors conclude that airway surface pH does not differ between cystic fibrosis and noncystic fibrosis subjects and therefore, cystic fibrosis transmembrane conductance regulator may not play a major role in airway surface pH in vivo.

Airways in cystic fibrosis are acidified: detection by exhaled breath condensate

Background: The loss of cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride conductance does not fully explain the diverse pathologies evident in patients with cystic fibrosis (CF). Bicarbonate (HCO3−) secretion...

The role of inflammation in the pathophysiology of CF lung disease.

Cystic fibrosis (CF) lung disease is characterized by a self-perpetuating cycle of airway obstruction, chronic bacterial infection, and vigorous inflammation that results in structural damage to the airway. CF patients have a predilection for infection with a limited spectrum of distinctive bacteria that initiate a vigorous inflammatory response which is more harmful than protective. The airway epithelial cell, which normally expresses the cystic fibrosis transmembrane conductance regulator (CFTR), directs the inflammatory response. Defects in CFTR are associated with increased production of pro-inflammatory mediators including IL-8, a potent neutrophil chemoattractant that stimulates the influx of massive numbers of neutrophils into the airways. These neutrophils are the primary effector cells responsible for the pathological manifestations of CF lung disease. Documented deficiencies in immunoregulatory molecules such as IL-10 likely contribute to the generation of the excessive and persistent inflammatory response. Since inflammation is a key contributor to the pathogenesis of CF lung disease, anti-inflammatory therapy must assume a larger role in CF until a cure is discovered. To date, attention has focused primarily on the therapeutic potential of systemic and inhaled corticosteroids and the non-steroidal anti-inflammatory drug (NSAID) ibuprofen. Development of new anti-inflammatory therapies that impact intracellular signaling pathways and cell-cell communication molecules likely will have the greatest impact on limiting the excessive production of the inflammatory mediators in the CF lung, thereby slowing the decline in lung function and improving survival.